P478: MOLECULAR CHARACTERIZATION OF EXTRAMEDULLARY LOCALIZATIONS IN ACUTE MYELOID LEUKEMIA (AML)
Authors: Giorgia Silvestrini, Tiziana Ottone, Rocco Giovanni Piazza, Serena Travaglini, Carmelo Gurnari, Francesco Marchesi, Anna Maria Nardozza, Emiliano Fabiani, Enrico Attardi, Mariadomenica Divona, Paola Ricci, Maria Antonietta Irno Consalvo, Sara Ienzi, Roberto Arcese, Annalisa Biagi, Luciano Fiori, Mariangela Novello, Alessandro Mauriello, Adriano Venditti, Lucia Anemona, Maria Teresa Voso
Journal: HemaSphere (2023)

Background: Extramedullary acute myeloid leukemia (EM-AML) is a rare manifestation of AML, associated with poor prognosis and shorter survival. Unlike solid tumors, the metastatic behaviors of blasts and the molecular mechanisms of EM tissue invasion by myeloid blasts have not been well characterized. Aims: To elucidate the molecular mechanisms underlying leukemia infiltration, we analyzed, by RNA sequencing, the expression profile of EM-AMLs localizations, as compared to that of paired BM blasts, isolated at the time of diagnosis or of relapse. The final goal was to identify predictive factors of EM-migration of AML cells, and possible druggable targets to be used to prevent or treat this prognostically adverse leukemic manifestation. Methods: Twelve patients (median age 60 years, range 53-75) affected by AML who developed an extramedullary AML localization at onset (N=5) or relapse (N=7), diagnosed at the Oncohematology Unit of Policlinico Tor Vergata in Rome, during the years 2019-2022, were included in the study. Paired samples of bone marrow (BM) and sarcoma tissue at the time of diagnosis and/or relapse were available in 7 EM-AML cases. For RNA-seq analysis, libraries were sequenced on the Illumina-platform with paired-end reads 150bp long, with a depth of 27 million cluster/sample. Statistical significance threshold for differentially expressed genes was set with an adjusted p-value < 0.1, and |log2(Fold Change)|≥ 2 to identify differentially expressed genes (DEGs) and pathways. These were then validated by q-RT-PCR. Functional experiments were then performed using siRNA and Metformin treatment, according to standard methods. Results: After comprehensive analysis of RNA-Seq data, the intersection of DAVID (v. 2021) and GSEA software (v. 3.0) output showed deregulation of the ECM-receptor interaction and Focal adhesion pathways as the most significantly enriched in EM sites, as compared to BM. Among identified pathways, 32 upregulated DEGs were common to both GSEA and DAVID analysis (Figure 1). Upregulation of these selected genes was confirmed by qRT-PCR in an independent EM-AML prospective cohort (n=3 pts). No differences in expression of cancer invasion pathway related genes were observed comparing BM-MNC (mononuclear cells) of EM-AML to that from healthy donors, suggesting that EM-AML cells may acquire specific invasive capabilities. To validate the transcriptome-based model of AML blasts to develop EM localizations, OCI-AML3 cells were transfected with siRNAs against TWIST1, a gene upregulated in EM-AML biopsies, which has an impact on cancer invasion as a result of dysregulation of EMT-related pathways. Forty-eight hours after siRNA transfection, we observed a significant reduction in cell proliferation as compared to non-treated cells. To confirm our model, cells were treated with Metformin, a drug able to repress TWIST1 expression and inhibit cancer invasion in several tumor models. After treatment with 20 mM Metformin for 48h, we confirmed reduction of EMT-related gene expression in OCI-AML3 cells. Finally, transwell assay showed a decreased cell migration and invasion after TWIST1 modulation. Summary/Conclusion: Our analysis suggests an involvement of processes closely associated with neoplastic cell invasion in EM-AML. Addition of Metformin to chemotherapy may help to reduce the development of EM-AML localizations.Keywords: Metastasis, Cell migration, Acute myeloid leukemia, RNA-seq